The present invention relates to a culture fluid concentrator used to concentrate a culture fluid cultivated previously which contains therein a specific useful substance such as physiologically active substance which has been produced by cells in a culture tank. Concentrated culture fluid is fed to a subsequent stage for extraction of said specific useful substance.
In general, production of specific useful substance by cell culture or microbe culture begins with growth of cells of microbes, then the specific useful substance is produced by these cells, followed by recovery and concentration of culture fluid, extraction of said specific useful substance, and further various stages such as decoloration and crystallization.
Presently adopted process for production of TRA (Human Tissue Plasminogen) or EPO (Erythropoietin) utilizing gene recombinant cells relies on such complicated process as mentioned above.
In such production process, an amount of the specific useful substance produced in a culture fluid is typically in the order of several mg/ml and subsequent stages to extraction of the specific useful substance from a culture fluid practically have relied on manual operation. Under the existing circumstances, only cultivation of cells and extraction of the specific useful substance have been mechanized.
Particularly in connection with the present invention, Japanese Disclosure Gazette No. 60(1985)-137280 discloses a technique according to a cell residual remover tank which is connected to a concentrator tank for cell products to effect a preliminary concentration by pressurization, decompression and circulation. A circulating continuous cultivation is also disclosed, by which culture fluid fed from a culture tank flows through a cell residual remover tank into a culture concentrating section, then added with a quantity of fresh culture ground and fed back again to the culture tank.
Japanese Disclosure Gazette No. 1986-199788 discloses a method for separation and concentration of microbe-produced substances utilizing hydrophobic porous membrane.
The culture fluid containing specific useful substance by cultivation in a culture tank usually contains therein, in addition to a large quantity of water, cell debris and protein. It also contains medium ground ingredients such as inorganic salts and serum, and impurities such as certain pharmacological effect inhibitors. Accordingly, the culture fluid is apt to be contaminated with microbes and various germs. Furthermore, the high protein concentration of culture fluid often causes bubble formation in various stages of treatment.
Particularly when protein secretion from cells occurs due to gene recombination, said protein concentration of the culture fluid necessarily increases and thus said phenomenon of bubble formation is inevitable. Though such culture fluid can be industrially produced with a productivity as high as several hundred liters per batch, efficient treatment of such large quantity of culture fluid has been impossible due to a limited filter area, when the conventional equipment for removal of impurities and for concentration of culture fluid is used to extract the specific useful substance from such a culture fluid,
When the concentrator tank is made of metallic material having no transparency, level detecting electrodes may be provided within the concentrator tank to monitor concentrate level. However, such electrodes have often malfunctioned due to bubbles formed within the tank and prevented the concentrator from being smoothly operated. Additionally, it has been difficult to wash said electrodes. The electrodes sometimes require manual washing which might result in loss of air-tightness being essential for concentrating system and cause contamination of the system components.
Particularly when said contamination is due to microbes or the like, the specific useful substance would be deactivated, correspondingly increasing the pharmacological inhibitors. The inventor has found that the contamination of the concentrating system with sundry germs or the like during production of EPO makes increase of pyrogen which is exothermic substance. A molecular weight of this pyrogen is so approximate to that of the specific useful substance that the pyrogen concentration increases as the specific useful substance concentration increases during a process of membrane concentration. In view of a fact that such pyrogen occurs various inhibiting actions, it is important to avoid such increase of pyrogen. To this end, it is essential to improve air-tightness of the concentrator system.